Environmental pollutants consist of a large quantity and variety of chemicals; many of these are toxic, environmental hazards that were designated in 1979 as priority pollutants by the U.S. Environmental Protection Agency. Microbial and enzymatic biodegradation is one method for the elimination of these pollutants. Accordingly, methods have been designed to treat commercial wastes and to bioremediate polluted environments via microbial and related enzymatic processes.
Unfortunately, many chemical pollutants are either resistant to microbial degradation or are toxic to potential microbial-degraders when present in high concentrations and certain combinations.
Haloalkane dehalogenase belongs to the alpha/beta hydrolase fold family in which all of the enzymes share similar topology, reaction mechanisms, and catalytic triad residues (Krooshof, et al., Biochemistry 36(31):9571-9580, 1997). The enzyme cleaves carbon-halogen bonds in haloalkanes and halocarboxylic acids by hydrolysis, thus converting them to their corresponding alcohols. This reaction is important for detoxification involving haloalkanes such as ethylchloride, methylchloride, and 1,2-dichloroethane, which are considered priority pollutants by the Environmental Protection Agency (Rozeboom, H., Kingma, J., Janssen, D., Dijkstra, B., Crystallization of Haloalkane Dehalogenase from Xanthobacter autotrophicus GJ10, J Mol Biol, 200 (3), 611-612 (1988)).
The haloalkane dehalogenases are produced by microorganisms that can grow entirely on chlorinated aliphatic compounds. No metal or oxygen is needed for activity: water is the sole substrate.
Xanthobacter autotrophicus GJ10 is a nitrogen-fixing bacteria that utilizes 1,2-dichloroethane and a few other haloalkane and halocarboxylic acids for growth (Rozeboom, et al., J Mol Biol, 200 3:611-612, 1988; Keuning, et al., J Bacteriol, 163(2):635-639, 1985). It is the most well-studied dehalogenase because it has a known catalytic reaction mechanism, activity mechanism and crystal-structure (Schanstra, et al., J Biol Chem, 271(25):14747-14753, 1996).
The organism produces two different dehalogenases. One dehalogenase is for halogenated alkanes and the other for halogenated carboxylic acids. Most harmful halogenated compounds are industrially produced for use as cleaning agents, pesticides, and solvents. The natural substrate of Xanthobacter autotrophicus is 1,2-dichloroethane. This haloalkane is often used in vinyl production.
Enzymes are highly selective catalysts. Their hallmark is the ability to catalyze reactions with exquisite stereo-, regio-, and chemo-selectivities that are unparalleled in conventional synthetic chemistry. Moreover, enzymes are remarkably versatile. They can be tailored to function in organic solvents, operate at extreme pH's and temperatures, and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates.
Enzymes are reactive toward a wide range of natural and unnatural substrates, thus enabling the modification of virtually any organic lead compound. Moreover, unlike traditional chemical catalysts, enzymes are highly enantio- and regio-selective. The high degree of functional group specificity exhibited by enzymes enables one to keep track of each reaction in a synthetic sequence leading to a new active compound. Enzymes are also capable of catalyzing many diverse reactions unrelated to their physiological function in nature. For example, peroxidases catalyze the oxidation of phenols by hydrogen peroxide. Peroxidases can also catalyze hydroxylation reactions that are not related to the native function of the enzyme. Other examples are proteases which catalyze the breakdown of polypeptides. In organic solution some proteases can also acylate sugars, a function unrelated to the native function of these enzymes.
The present invention exploits the unique catalytic properties of enzymes. Whereas the use of biocatalysts (i.e., purified or crude enzymes, non-living or living cells) in chemical transformations normally requires the identification of a particular biocatalyst that reacts with a specific starting compound, the present invention uses selected biocatalysts and reaction conditions that are specific for functional groups that are present in many starting compounds.
Each biocatalyst is specific for one functional group, or several related functional groups, and can react with many starting compounds containing this functional group.
The biocatalytic reactions produce a population of derivatives from a single starting compound. These derivatives can be subjected to another round of biocatalytic reactions to produce a second population of derivative compounds. Thousands of variations of the original compound can be produced with each iteration of biocatalytic derivitization.
Enzymes react at specific sites of a starting compound without affecting the rest of the molecule, a process which is very difficult to achieve using traditional chemical methods. This high degree of biocatalytic specificity provides the means to identify a single active compound within the library. The library is characterized by the series of biocatalytic reactions used to produce it, a so called “biosynthetic history”. Screening the library for biological activities and tracing the biosynthetic history identifies the specific reaction sequence producing the active compound. The reaction sequence is repeated and the structure of the synthesized compound determined. This mode of identification, unlike other synthesis and screening approaches, does not require immobilization technologies, and compounds can be synthesized and tested free in solution using virtually any type of screening assay. It is important to note, that the high degree of specificity of enzyme reactions on functional groups allows for the “tracking” of specific enzymatic reactions that make up the biocatalytically produced library.
Many of the procedural steps are performed using robotic automation enabling the execution of many thousands of biocatalytic reactions and screening assays per day as well as ensuring a high level of accuracy and reproducibility. As a result, a library of derivative compounds can be produced in a matter of weeks which would take years to produce using current chemical methods. (For further teachings on modification of molecules, including small molecules, See PCT/US94/09174, herein incorporated by reference in its entirety).
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.